Method and apparatus for separating molecules using micro-channel

ABSTRACT

A method for simply and efficiently separating substances by utilizing a specific flow behavior in a non-turbulent flow, i.e. a laminar flow, in a micro-channel is disclosed. A mixed solution containing at least two kinds of solute molecules which are different from each other in molecular weight and/or molecular sharp, or at least two kinds of solutions containing their respective solute molecules are flowed into a micro-channel to form a non-turbulent flow. A physical action is given to each molecule by changing the state of flow, thereby causing different behaviors among the different solute molecules. By utilizing this behavior difference, molecules of a specific kind are gathered in a specific region in the channel for separation.

TECHNOLOGICAL FIELD

The present invention relates to a novel method for separating moleculesor agglomerates of molecules such as cells by molecular species from amixture of two kinds or more of molecules or, more particularly, to anovel method for separating different molecules or agglomerates ofmolecules by utilizing differences in the behaviors between two kinds ormore of the solute molecules in a solution which are brought about byeffecting changes in a non-turbulent flowing condition caused in a microflow channel as well as an apparatus for carrying out the same.

BACKGROUND TECHNOLOGY

In the preparation of a chemical substance, it is indispensable, in mostcases, to undertake separation and purification of the product as thefinal step. In continuously conducting a reaction for the preparation ofa desired chemical substance, it is also a necessary treatment toseparate and purify the intermediate product as the intermediate step inorder to accomplish a higher reaction rate and higher efficiency.

A great variety of methods are known heretofore as the means for suchseparation and purification including solvent-extraction methods using asolvent, fractionating precipitation methods from a solution, filtrationmethods through a filtering material, dialysis methods through apermeable membrane, fractionating distillation methods utilizing thedifference of the boiling points, zone-melting methods suitable for thepurification of single crystals, electrophoresis methods,chromatographic methods and so on and they are utilized as adequatelyselected depending on the object of separation thereof.

However, no methods for carrying out separation or purification betweenmolecular species by utilizing laminar flows as formed in a micro flowchannel are known heretofore.

DISCLOSURE OF THE INVENTION

The present invention has been completed with an object to provide amethod for separation of substances with easiness and efficiency byutilizing the characteristic action of the flowing behaviors under anon-turbulent condition, or namely, a laminar-flow condition within amicro flow channel as well as an apparatus suitable for carrying out thesame.

The inventors have continued extensive investigations with regard to therelationship between a non-turbulent flowing condition within a microflow channel and substance molecules therein and, as a result, havearrived at a discovery that, changes in the non-turbulent flowingcondition accompany addition of the characteristic action on the solutemolecules existing in a solution under a non-turbulent flowing conditionin which the action depends on a mass of molecule or, namely, amolecular weight or a molecular configuration so that, by utilizing thesame, it is possible to separate and purify, with easiness, two kinds ormore of molecules having different molecular weights or molecularconfigurations leading to completion of the present invention on thebase of this discovery.

Namely, the present invention relates to a method for separation ofmolecules characterized by comprising the steps of: passing a mixedsolution containing at least two kinds of solute molecules havingdifferent molecular weights and/or different molecular configurationseach from the other or at least two kinds of solutions each containingthe respective solute molecules independently from the other through amicro flow channel under a non-turbulent flowing condition; adding aphysical action to each of the molecules by modifying the flowingcondition thereof thereby to cause differences in the behaviors betweendifferent kinds of the solute molecules brought about by the saidaction; utilizing the same to cause localization of the molecules of aspecified kind only in a specified zone within the flow channel; andseparating the same as well as an apparatus for separation of moleculessatisfactory for carrying out the method.

The “non-turbulent flowing condition” here implied means a condition inwhich parallel flows in a definite direction are formed withoutoccurrence of a turbulent flow in every portion within a cross sectionof the flow.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a plan view showing tracks obtained in Example 1.

FIG. 2 shows cross sectional views of the flow channel in the front andin the rear of the bent portions in Example 2.

FIG. 3 is a plan view of the micro flow channel used in Example 3.

FIG. 4 is an explanatory drawing of the main part of the propertydetecting sensor used in Examples 3 and 4.

FIG. 5 is a bar chart showing the results of Example 3.

FIG. 6 is a plan view of the micro flow channel used in Example 4.

FIG. 7 is a bar chart showing the results of Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION

The micro flow channel used in the method of the present invention canbe constituted of a capillary tube made of an inactive material or canbe provided in the form of a groove on a base plate made of an inactivematerial. The inactive material implied here is a material, whichexhibits no reactivity with the solvents, solutes and compounds producedby the reaction, as exemplified, for example, by glass, quartz orsilica, Si/SiO₂, magnesia, zirconia, alumina, apatite, silicon nitrideand ceramic materials including oxides, carbides, nitrides, borides,silicides and the like of metals such as titanium, aluminum, yttrium andtungsten.

In addition, any metals, plastics and the like can also be used providedthat they are inactive materials. With respect to the form of the baseplate, it can usually be a flat plate but, if so desired, those havingan arch-wise form, spherical form, granular form and others can be used.

The micro flow channel is prepared by engraving as a groove in a size of1 to 1000 μm or, preferably 50 to 500 μm width and depth, or formed as acapillary tube having a comparable size. It is desirable to properlyselect the size depending on the viscosity and flow rate of the solutiontaken into consideration the hydrodynamic variables such as the Reynoldsnumber and the like, and others. The length of this micro flow channelis, though not particularly limited provided that it is selected,corresponding to the kinds and conditions of solute molecules to beseparated, in the range, usually, from 100 to 1000 mm.

Such a micro flow channel can be a commercial capillary tube used assuch or can be prepared by engraving, on a base plate of an inactivematerial, by a mechanical means using a machine tool such as amicrodrill or, alternatively, by engraving with the photolithographyused in the manufacture of semiconductor integrated circuits and othersfollowed by adhesive bonding of another base plate thereto.

When a fluid such as a liquid is introduced to flow in such anextra-fine micro flow channel, the liquid flows straightly in a definitedirection or, namely, in the direction of the flow channel under anon-turbulent flowing condition. Such an extra-fine flow channel hascharacteristics of a short diffusion distance of solute molecules,relatively large contacting area with the wall surface, a large gradientof the flow velocity within a cross section of the flow channel, andothers.

When such a micro flow channel has a portion not straightly linear likea bent portion, even though the non-turbulent flowing condition can bemaintained, the solvent molecules and solute molecules contained thereinare under physical actions by the centrifugal force and force of inertiaat the bent portions and centrifugal force, force of inertia orcollision and rebounding at the wall surface in the bent portionsdepending on differences in the configuration of the micro flow channel,velocity of the flow, steric structure and molecular weight of themolecules and the like. And, these actions cause a secondary flow of thesolution within the flow channel or, namely, the flow having componentsin the direction of the flow channel and the perpendicular direction.

In the present invention, separation of the objective substances isperformed by means of the molecular screening effect obtained byutilizing one or a plurality of these actions. Among the aforementionedplurality of the physical actions including the centrifugal force, forceof inertia, secondary flows and others, the type of the action and theextent of the influence caused thereby depend on the kind of the solutemolecules as the objective of separation.

For example, a centrifugal force acts at the bent portion of the flowchannel so as to attract the heavier molecules more toward outside.Since the strength of the force depends on the weight of the solutemolecules and the curvature of the curve, separation of the objectivesolute molecules can be accomplished by the utilization of this physicalphenomenon. Furthermore, while the solute molecules in the solution areunder continuous impingement of the solvent molecules and the frequencyof such impingements depends on the conformation of the solutemolecules, the conformation of the solute molecules is also an importantfactor in conducting separation in addition to the molecular weight andcurvature of the bent portion so that separation can be effected on thebase of the conformation of the solute molecules.

When a solution flows along a portion of the flow channel not straightlylinear as in the bent portions, furthermore, a secondary flow isgenerated within the flow channel by means of the centrifugal force andthe force of inertia acting on the solvent molecules as generated there.Accordingly, still higher performance of separation can be accomplishedby jointly utilizing this phenomenon and the aforementioned force actingon the solute molecules.

In this way, localization of specified solute molecules can beaccomplished in specified regions within the flow channel and such astate of localization can be maintained insofar as a non-turbulent flowcondition is realized within the micro flow channel so that a solute asdesired can be selectively taken out by means of a control of the flowchannel structure, i.e. by forming the outlet structure of the flowchannel to be branched from the aforementioned portion of localization.

In this way, according to the method of the present invention,localization of giant molecules such as proteins and the like can beefficiently accomplished based on the molecular weight and stericstructure thereof by means of the molecular screening effect utilizing amicro flow channel thereby to carry out function analyses of proteinsconveniently and rapidly at low cost.

According to the method of the present invention, the object can beachieved within a remarkably short time with easiness as compared withgel electrophoresis conventionally used for the same object. And, anadditional advantage is obtained that objective substances such asproteins and the like alone can be selectively taken out. Furthermore,it is possible to continuously introduce the solution so that handlingof a large number of samples is practicable.

According to the method of the present invention, It is also possible toutilize as an analytical means such as quantitative determination andthe like by measuring the amounts of the solute molecules localized in apart within the flow channel.

When, in the method of the present invention, a mixed solutioncontaining two kinds or more of different molecules is introduced or twokinds or more of solutions each containing different molecules areindividually introduced, as keeping contact each with the other, intothe micro flow channel, the mixed solution forms two kinds or more offlows having respectively different molecular concentrations or thosesolutions flow in a condition with formation of an interface withoutbeing intermixed each with the other. In the latter case, a complex isformed between the solute molecules of those solutions on this interfaceprovided that they have specific affinity therebetween and, in the casesof DNAs where the base sequences are complementary or a protein and asubstrate with a specific interaction therebetween, for example, changesare caused in the molecular weights or in the molecular configurations.Thereby, separation can be conducted by causing selective localizationof the thus formed complex alone or analyses are conducted by utilizingthe same.

In the method of the present invention, the solution can be introducedto the micro flow channel, for example, by carrying out manually with aninjector while it is advantageous to undertake a mechanical means suchas a syringe pump and the like to automatically carry out under controlof the liquid feed rate, liquid feed pressure and others.

In the method of the present invention, separation of the objectivemolecules can be conducted by virtue of the simple procedure merely topass a solution through a micro flow channel to accomplish separationwithin a greatly shortened time as compared with the prior artseparation methods by utilizing the molecular screening effect and,furthermore, it is an analytical method of wide applicability that agreat variety of separations can be accomplished by modifying thepassing conditions and, in addition, there can be obtained an advantageenabling a high-performance separation such as multiple-stage separationand others by way of the design of the flow channel and high-precisionseparation by way of temperature control.

In the following, the best mode for carrying out the present inventionis described by way of examples although the present invention is neverlimited by these examples in any way.

EXAMPLE 1

The experiments were undertaken for accomplishing movement of solutemolecules in a solution, when a solution is passing over a bent portionof a micro flow channel, toward the outward direction of the bentportion of the flow channel by the centrifugal force or other physicalactions.

FIG. 1 is a plan view of the track drawn by the duplex DNA moleculeshaving 20 pairs of bases with a molecular weight of 12000 found in thecenter portions when an aqueous solution was passed through a micro flowchannel having a width of 360 μm and a depth of 200 μm in the U-shapedcross section at a velocity of 10 mm/second. The bent portion of theflow channel here had a radius of curvature of 1 mm.

It is understood from this figure that the DNA molecules formerly foundon the centerline of the flow channel had moved toward the outwarddirection of the flow channel curve during turning of the curve by aphysical action such as a centrifugal force and others. Since the valueof this physical action depends on the molecular weight, molecularconfiguration and others, it is possible to freely modify the extent ofthe molecular screening effect by controlling the liquid-flowingconditions.

EXAMPLE 2

Experiments were undertaken for observation of the condition ofinterface deformation when a solution was flowing in the bent portion ofa micro flow channel by directly taking a picture of a cross section ofthe flow channel by means of a confocal laser scanning microscope.

FIG. 2 is a cross sectional view of a flow channel in the front of andin the rear of bent portions under flowing of an aqueous solutioncontaining fluorescein as a fluorescent dye and pure water free from thesame by keeping contact therebetween through an S-shaped micro flowchannel having a width of 360 μm and a depth of 200 μm at a velocity of10 mm/second.

As is understood from this figure, the interface of the two solutionsmoved in the outward direction by the movement of the inner sidesolution under the physical action received at the bent portions. Thismeans that a secondary flow is generated within the flow channel by theoutward movement of the solvent also under a physical action such as acentrifugal force and others. Accordingly, separation of substances canbe performed in an increased efficiency by the combination of themolecular screening effect on the solute molecules alone and thisaction.

EXAMPLE 3

Experiments were undertaken for separation and analysis by utilizing themolecular screening effect caused by the weight increase due to theformation of a complex between two solutions flowing in a micro flowchannel as compared with the weight before complex formation. Detectionwas performed here for duplex DNAs formed sequence-selectively.

As the probe DNA, was prepared a DNA fragment having fluorecein of thefluorescent substance introduced to the 5′ terminal as follows:F-(5′)-AGGCTGCTCCCCGCGTGGCC-(3′) (wherein F is fluorecein).

As the sample DNAs were prepared two kinds of DNA fragments as follows:

(5′)-GGCCACGCGGGGAGCAGCCT-(3′) (referred to as the sample 1hereinafter). (5′)-AAAAAAAAAAAAAAAAAAAA-(3′) (referred to as the sample2 hereinafter).

Four solutions as a total were prepared including a solution containingno DNA fragments (referred to hereinafter as the blank solution) alongwith the three kinds of the DNA fragment solutions. The solutions had asolution composition of 1 pmol/μl of DNA, 5 mM phosphate buffer solution(pH 7.0) and 50 mM of sodium chloride. Three combinations includingthose of the probe DNA solution and the sample 1 solution, the probe DNAsolution and the sample 2 solution and the probe DNA solution and theblank solution were each introduced to the micro flow channel systemwith a configuration as shown in FIG. 1. A liquid-introducing rate wasset to be 20 μl/min.

FIG. 3 shows a plan view of a micro flow channel having four bentportions. This flow channel has the same cross section as that inExample 2. FIG. 4 is an explanatory drawing showing the microscopeportion including the fluorescence detector or, namely, the propertydetecting sensor. And, irradiation was made with a beam of an argon gaslaser of 488 nm to the test sample flow channel side at the position Ain the micro flow channel to cause emission of fluorescence andcomparison was made for the intensities thereof which were detected bythe microscope. The results are shown in FIG. 5 as a bar chart. Thesevalues are the average values obtained by ten-times measurements of thefluorescent intensities (in an arbitrary unit) and the ranges of thestandard deviations are indicated with error bars. It is understood fromthis figure that a particularly high fluorescence response can beobtained only in the case of the sample 1 having a base sequencecomplementary with the probe DNA fragments as comparison was made withthe other two controls.

It is understood from these results that those having specificinteractions or, namely base-sequence complementary ones are convertedinto a heavy complex by forming a duplex chain at the interface andmoved toward the test sample flow channel side by the molecularscreening effect at the bent portions.

The results of the measurements have a variation coefficient of around3% to indicate analyzability with very high reproducibility.

EXAMPLE 4

Since, in the method of the present invention, the efficiency ofseparation depends on the molecular weight or the molecular size, it ispossible to construct an instrument for the determination of the lengthof the sample DNAs from the fluorescence intensity by applying this factto the means of Example 3. In this Example, the experiments areindicated.

The probe DNA used here was the same one as used in Example 3 and thesample DNAs prepared here included Sample 1 in Example 3 and thoselisted below.

(5′)-CACGCGGGGA-(3′) (referred to as the sample 3 hereinafter).

(5′)-CCACGCGGGGAGCAG-(3′) (referred to as the sample 4 hereinafter).

(5 ′)-CCGGTGTAGGAGCTGCTGGTGCAGGGGCCACGCGGGGAGCAGCCTCTGGCATTCTGGGAGCTTCATCTGG-(3′) (referred to as the sample 5 hereinafter).

Solutions were prepared each having a composition of 1 pmol/μl of DNA, 5mM phosphate buffer solution (pH 7.0) and 5 mM of sodium chloride byusing these five kinds of the DNA fragments. Four combinations includingthose of the probe DNA solution and the sample 1 solution, the probe DNAsolution and the sample 3 solution, the probe DNA solution and thesample 4 solution and the probe DNA solution and the sample 5 solutionwere each introduced to the micro flow channel system with aconfiguration as shown in FIG. 6. In this case, a liquid-introducingrate was set to be 40 μl/minute and the temperature was 35° C.

FIG. 6 is a plan view of the micro flow channel bent eight-fold as usedin this Example. This flow channel had a cross sectional profile whichwas the same as in Example 3. Irradiation with a 488 nm argon gas laserbeam was conducted at each of the test sample flowing channel side andthe probe flowing channel side on the spot B in the micro flow channelto cause fluorescence emission and to measure the intensity thereof bymeans of a fluorescence detector making evaluation by way of theintensity ratio of the two fluorescences. The results are shown in FIG.7 as a bar chart. These values are average values obtained by ten-timesmeasurements of the fluorescence intensities and the ranges of thestandard deviations are indicated with error bars. As is understood fromthis figure, the response obtained corresponded to the length of thesample DNA fragments taken as the objective of detection. Thus, it isnow possible by using this device to know the size of an unknown sampleDNA fragment from the ratio of the fluorescence intensities.

INDUSTRIAL UTILIZABILITY

The present invention is applicable to the maneuvers for separation ofchemical substances in general or, particularly, satisfactory forseparation of high molecular-weight substances such as, for example,macromolecular substances, DNAs, proteins and the like.

1. A method for separation of molecules characterized by comprising thesteps of: passing a mixed solution containing at least two kinds ofsolute molecules having different molecular weights and/or differentmolecular configurations each from the other or at least two kinds ofsolutions each containing the respective solute molecules independentlyfrom the other through a micro flow channel under a non-turbulentflowing condition; adding a physical action to the respective moleculesby modifying the flowing condition thereof thereby to cause differencesin the behaviors between different kinds of the solute molecules broughtabout by the said action; and utilizing the same to cause localizationof the molecules of a specified kind only in a specified zone within theflow channel for separation.
 2. The method for separation of moleculesdescribed in claim 1 in which the physical action is selected from asecondary flow, a centrifugal force and a force of inertia.
 3. Themethod for separation of molecules described in claim 1 in which thedifference in the molecular weights or in the molecular configurationsbetween different kinds of solute molecules is amplified by forming acomplex of the molecules to be separated along the interfacial surfacebetween the respective solutions flowing under the non-turbulent flowingcondition.
 4. An apparatus for separation of molecules constituted of: asubstrate plate provided with a micro flow channel engraved thereinhaving one or at least two bent portions, of which one end serves for asample inlet port and the other end serves for a sample outlet port; anda sensor means for detection of physical properties disposed incorrespondence to the flow at the inward side or at the outward side ofthe bent portions.